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Simultaneous Determination of Reactive Oxygen and NitrogenSpecies in Mitochondrial Compartments of Apoptotic HepG2 Cellsand PC12 Cells Based On Microchip Electrophoresis−Laser-InducedFluorescenceZhenzhen Chen,† Qingling Li,† Qianqian Sun,† Hao Chen,‡ Xu Wang,† Na Li,† Miao Yin,† Yanxia Xie,†

Hongmin Li,† and Bo Tang*,†

†College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes, Ministry ofEducation, Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education, ShandongNormal University, Jinan 250014, People’s Republic of China‡Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio 45701,United States

*S Supporting Information

ABSTRACT: Determination of intracellular bioactive specieswill afford beneficial information related to cell metabolism,signal transduction, cell function, and disease treatment. In thisstudy, the first application of a microchip electrophoresis−laser-induced fluorescence (MCE−LIF) method for con-current determination of reactive oxygen species (ROS) andreactive nitrogen species (RNS), i.e., superoxide (O2

−•) andnitric oxide (NO) in mitochondria, was developed usingfluorescent probes 2-chloro-1,3-dibenzothiazolinecyclohexene(DBZTC) and 3-amino,4-aminomethyl-2′,7′-difluorescein(DAF-FM), respectively. Potential interference of intracellulardehydroascorbic acid (DHA) and ascorbic acid (AA) for NOdetection with DAF-FM was eliminated through oxidation ofAA with the addition of ascorbate oxidase, followed by subsequent MCE separation. Fluorescent products of O2

−• and NO,DBZTC oxide (DBO), and DAF-FM triazole (DAF-FMT) showed excellent baseline separation within 1 min with a runningbuffer of 40 mM Tris solution (pH 7.4) and a separating electric field of 500 V/cm. The levels of DBO and DAF-FMT inmitochondria isolated from normal HepG2 cells and PC12 cells were evaluated using this method. Furthermore, the changes ofDBO and DAF-FMT levels in mitochondria isolated from apoptotic HepG2 cells and PC12 cells could also be detected. Thecurrent approach was proved to be simple, fast, reproducible, and efficient. Measurement of the two species with the method willbe beneficial to understand ROS/RNS distinctive functions. In addition, it will provide new insights into the role that bothspecies play in biological systems.

Reactive oxygen species (ROS), such as superoxide (O2−•)

and hydrogen peroxide (H2O2), and reactive nitrogenspecies (RNS), such as nitric oxide (NO) and peroxynitrite(ONOO−), are important players in a multitude ofpathophysiological conditions.1 They have apparently dichot-omous effects under certain circumstances. Appropriate amountof these species is essential to the immune response and manyphysiological signal transduction pathways; however, whenoverproduced, their chemical reactivity can lead to toxicity andtissue injury.2 In addition to their respective biological activities,it is also important to investigate ROS and RNS relationships incomplex biological situations. For example, the interactionbetween signaling molecule NO (produced endogenously bythe action of NO synthase, NOS) and O2

−• withinmitochondria is of pathological significance and is also apotential mechanism for the regulation of mitochondrial

function.3 On one hand, the inhibition of cytochrome coxidase4 situated on the inner membrane of mitochondria byNO leads to increased superoxide production by respiratorycomplexes I and III. The overproduced superoxide can beeffectively converted to more oxidizing hydrogen peroxide(H2O2) by mitochondrial superoxide dismutase (SOD). On theother hand, superoxide can inactivate NO action at a nearlydiffusion-controlled speed to form peroxynitrite (ONOO−),5 astrong oxidant that can react with a vast number of otherbiomolecules to cause cell damage. Considering the importantimplication mechanism of mitochondrial O2

−• and NO,

Received: September 22, 2011Accepted: May 2, 2012Published: May 2, 2012

Article

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© 2012 American Chemical Society 4687 dx.doi.org/10.1021/ac300255n | Anal. Chem. 2012, 84, 4687−4694

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together with their interaction for biochemical regulation, it isof interest to examine the production of O2

−• and NO inmitochondria simultaneously.To date, O2

−• and NO are often detected individually byvarious analytical techniques, such as electron spin resonance(ESR),6 electrochemistry with microelectrodes,7,8 chemilumi-nescence,9,10 flow cytometry,11,12 and fluorimetry,13 which havebeen successfully applied to estimate the production of eitherspecies in biological systems. However, the data obtained fromtwo different situations could not track the contents of thesetwo species and their relationships at the same time point. Apotent method for simultaneous analysis of the two species isstill urgently needed. Microchip electrophoresis coupled withlaser-induced fluorescence (MCE−LIF) detection is a powerfultool for simultaneous measurement of complex ingredients,14

with many attractive benefits such as chip size reduction,reagent consumption reduction, and analysis time decrease,which can reduce overall cost. The combination of differentfunctions on a single microchip is also beneficial to maintain acompletely closed system with merits of automation,contamination, human intervention reduction, and errorelimination. In addition, the high aggregation of the laserbeam is fit for samples with limited volume, which can greatlyenhance the detection sensitivity. Diversified bioactive speciessuch as DNA,15 protein,16 virus,17 ROS,18,19 GSH,20 etc. havebeen analyzed with the method. From the perspective oftechnology, it is feasible to detect mitochondrial O2

−• and NOconcurrently.Recently, we have successfully established O2

−• and H2O2,21

H2O2 and GSH22 simultaneous determination platforms usingthe MCE−LIF method associated with ideal fluorescent probesand realized concomitant measurement of them in cell extractsand subcellular compartments. Undoubtedly, mutual interplayof bioactive species has become an area of intense research forthe function of them in intricate biological processes.23 As forcellular O2

−• and H2O2, the short lifetime of O2−• will result in

its transformation toward H2O2 and subsequent H2O2accumulation; therefore, concurrent determination of them issignificant for understanding their roles in cellular signaltransduction.21 During apoptosis, mitochondrial GSH concen-tration decreases while H2O2 concentration increases, reflectingthe varying redox conditions in mitochondria; simultaneousdetection of H2O2 and GSH during the apoptotic course mayhelp further comprehend the apoptosis mechanism of differentcell types.22 O2

−• and NO belong to different bioactive species,but their biological activities could be cancelled out by formingperoxynitrite. Due to the prompt conversion of O2

−•, theexistence of mitochondrial nitric oxide synthase (mtNOS), andother biological activities associated with them, the levels ofO2

−• and NO in mitochondria during different pathophysio-logical conditions are difficult to predict, especially duringapoptosis. It is well-known that the concentration range ofbioactive species is a key determinant of their biologicalfunctions.24,25 Therefore, concurrent measurement of bothspecies is important for understanding their precise roles inrelated research.Herein, we have developed an MCE−LIF method for

simultaneous analysis of superoxide and nitric oxide usingfluorescent probes of 2-chloro-1,3-dibenzothiazolinecyclohex-ene (DBZTC)26 and 3-amino,4-aminomethyl-2′,7′-difluorescein(DAF-FM).27,28 Interference from dehydroascorbic acid(DHA) and ascorbic acid (AA)27 for DAF-FM usage waseliminated by a specific enzymatic reaction28 and then

separation of various derivative products to complete NOdetermination.29 The optimum conditions including buffertype, concentration, and separating electric field intensity weredetermined. Using the MCE−LIF assay, we determinedmitochondrial O2

−• and NO in HepG2 cells and PC12 cells.We then attempted to monitor the changes of O2

−• and NOlevels in mitochondria during cell apoptosis, since it issuggested that the mitochondria plays a central role in responseto cell survival, while mitochondrial O2

−• and NO arerecognized as important regulators of apoptotic pathways.30

To induce cell apoptosis, resveratrol that exerts antiproliferativeand apoptotic actions on cancer cell lines, and amyloid β (Aβ)that is a major factor in the pathogenesis of Alzheimer’s disease(AD), were introduced to HepG2 cells and PC12 cells,respectively. The method characteristics, time course change ofmitochondrial DBO and DAF-FMT levels of the two cell typesduring apoptosis were studied. The results confirmed thefeasibility of the method, which provided new chances forunderstanding both species’ pathobiological and therapeuticbasis for diverse diseases.

■ EXPERIMENTAL SECTION

Reagents. All chemicals and solvents used were of analyticalgrade. Water was purified with a Sartorius Arium 611 VFsystem (Sartorius AG, Germany) to a resistivity of 18.2MΩ·cm. DBZTC was synthesized in our laboratory. 3-Amino,4-aminomethyl-2′,7′-difluorescein, diacetate (DAF-FMDA) and diethylamine NONOate (DEANO) were obtainedfrom Sigma-Aldrich Chemicals (St. Louis, MO, U.S.A.). Stocksolutions of DBZTC and DAF-FM DA (5.0 mM) wereprepared in dimethyl sulfoxide (DMSO) and stored at −20 °Cin darkness separately. For further use, the working solutions ofDBZTC and DAF-FM DA were prepared by diluting the stocksolutions. The preparation and purification of DBZTC oxide(DBO) followed the procedure from ref 26. Its stock solutionwas prepared by dissolving DBO in DMSO to 1 mM. Tris,borate, HEPES, and phosphate buffer used for electrophoreticmigration were prepared by dissolving appropriate amount ofTris, Na2B4O7, HEPES, and NaH2PO4 in ultrapure water,respectively. The required pH was adjusted by adding theappropriate amount of HCl or NaOH. Ascorbate oxidase fromCucurbita sp. (AO, lyophilized powder, Sigma) was dissolved in100 mM PBS (containing 0.5 mM EDTA, pH 5.6) before use.Due to the instability of dehydroascorbic acid (DHA), itssolution was freshly made in a nitrogen-purged buffer beforeuse.For cell treatment, a 5.0 mM resveratrol (Sigma) was

prepared in DMSO. Subsequent dilutions were done in culturemedium. Amyloid β25−35 peptide (Aβ25−35, Sigma) wasdissolved at 2 mM in sterile deionized water and kept frozenuntil use. The peptide was incubated at 37 °C for 2 days toprepare aggregated Aβ25−35 fibrils.31 The solution was thendiluted to the required concentration with serum-free DMEM.Aβ25−35 was used at 30 μM in all procedures unless otherwisestated.

DAF-FM and DAF-FMT Solution Preparation. DAF-FMDA is a cell-permeable fluorescent probe for the detection ofnitric oxide intracellularly, which is hydrolyzed to DAF-FM byintracellular esterases. As for nitric oxide quantification andmitochondrial determination, DAF-FM was prepared justbefore use by adding the appropriate amount of esterase(Sigma) to the desired concentration of DAF-FM DA solution.

Analytical Chemistry Article

dx.doi.org/10.1021/ac300255n | Anal. Chem. 2012, 84, 4687−46944688

A NO donor, DEANO, produces two molecules of NOeach.32 The delivery of NO can easily be controlled bypreparing moderately basic solutions of the NONOate andthen lowering the pH to initiate NO generation. Stock solutionof NO (12.9 mM) was prepared by dissolving 1.0 mg ofcrystalline DEANO in 1 mL of 0.01 M sodium hydroxidesolution. Working solutions were prepared by dilution of thealkaline NONOate solution in 0.1 M deoxygenated phosphatebuffer (pH 7.4).Standard solutions of DAF-FMT were obtained by

combining different concentrations of NO solutions with 2μM DAF-FM; then the mixed solutions were allowed to reactfor 40 min at 37 °C.Cell Culture and Treatment. HepG2 cells (American

Type Culture Collection, Manassas, VA) were grown in cellculture media and incubated at 37 °C in a humidifiedatmosphere of 5% CO2 and 95% air. The cell culture mediumwas RPMI-1640 (Hyclone, U.S.A.) supplemented with 10%newborn calf serum (Gibco, Invitrogen), 1% penicillin, and 1%streptomycin.PC12 cells, obtained from the Chinese Type Culture

Collection (Shanghai Institute of Cell Biology, ChineseAcademy of Science, China), were grown in cell culturemedia and incubated at 37 °C in a humidified atmosphere of5% CO2 and 95% air. The cell culture medium was high-glucose DMEM (Hyclone, U.S.A.) supplemented with 10%newborn calf serum (Gibco, Invitrogen), 1% penicillin, and 1%streptomycin. Cell viability was determined by the trypan-blueexclusion assay.When cells were in a logarithmic growth phase, resveratrol at

a final concentration of 100 μM was administered into theHepG2 cell culture medium. As for the Aβ25−35-inducedapoptosis, a final concentration of 30 μM was administeredinto the PC12 cell.Preparation of Mitochondrial Extracts. Before and after

treatment of cells with apoptosis-inducing reagent, mitochon-dria were prepared by differential centrifugation using acommercially available Beyotime mitochondria isolation kit(Beyotime Inst. Biotech, Haimen, China) with the aid of adounce homogenizer. After centrifugation (Sigma, 3K15,Germany), the mitochondria pellet was suspended in uptakebuffer (70 mM sucrose, 1 mM KH2PO4, 5 mM sodiumsuccinate, 5 mM HEPES, 220 mM mannitol, 0.1 mM EDTA,pH 7.4) to a final concentration of 0.5 mg/mL protein. Allsteps were performed below 4 °C, and the isolatedmitochondrial sample was kept on ice until being used in theexperiments.Mitochondria Treatments. For superoxide depletion,

Tiron, an SOD mimetic that scavenges superoxide, was addedinto the mitochondria extract to a final concentration of 1 mM.Depletion of nitric oxide was performed by addition of 10 μMhemoglobin to the mitochondrial extract. When needed, themitochondrial extracts were treated with 5 μM rotenone toinhibit mitochondiral respiration. Then the mitochondrialsolutions were determined according to the microfluidicelectrophoresis procedure.Protein Quantification. To measure the protein concen-

tration in a mitochondrial extract, the dye-binding assay ofBradford was used with Bradford protein assay kit (BeyotimeInst. Biotech, Haimen, China). Absorbance at 595 nm wasmeasured with a microplate reader RT6000 (Rayto, American).Microfluidic System. The schematic diagram of the

experimental setup is shown in the Supporting Information,

Figure S1. The microfluidic system consisted of a glassmicrochip, a laser-induced fluorescence detector (LIFD), aversatile programmable eight-path-electrode power supply(PEPS), a data acquisition, and a personal computer. Themicrochip with a cross design was made in house. The channelcross section was close to a rectangle structure (70 μm width×25 μm depth). Four reservoirs were the same columnstructure with 3 mm diameter and 1.5 mm depth. The glassmicrochip assembly was mounted on the X−Y−Z translationalstage of the LIFD. The laser detection point lay 30 mmdownstream from the cross, C, in the separation channel. ThePEPS was used for sample injection and MCE separation. Theconnecting interface between the PEPS and the chip wasrealized by dipping the four Pt electrodes (0.5 mm) of thePEPS into the four reservoirs of the microfluidic chip,respectively.The LIFD and PEPS were both made in house. A low-noise

semiconductor double-pumped solid-state laser (MLLIII-473nm/20 mW, Changchun Xinchanye Guangdianjishu Co. Ltd.,China) was reflected using a dichroic splitter (Omega OpticalInc., Brattleboro, VT, U.S.A.) and focused by a 40× objectivelens (Leica instrument Co. Ltd., Germany) into the separationchannel. The emitted fluorescence was collected by the sameobjective and penetrated through the same dichroic splitter, aband-pass filter, a focusing lens, a 500 μm pinhole, a 525 ± 5nm narrow band filter, and was finally detected by an R928photomultiplier (PMT, Hamamatsu, Japan). Signal output ofthe PMT passing through the I/V converters (OPA128, BBInc., U.S.A.) was sampled using a CT-22 data acquisition card(the sampling frequency, 20 Hz, Shanghai Qianpu Shuju Co.Ltd., China). The computer incorporated with a program wasused to control the PEPS and data acquisition.

Microfluidic Electrophoresis Procedure. The micro-channels were rinsed sequentially with 0.1 M NaOH andultrapure water for 10 min, respectively, before being flushedwith electrophoresis buffer for 5 min. Prior to the MCEseparation, sample waste (SW), buffer (B), and buffer waste(BW) reservoirs were all filled with 10 μL of electrophoresisrunning buffer, and sample reservoir (S) was filled with 10 μLof sample solution. The electrokinetic pinched sample injectionand zone electrophoresis separation were controlled by thevoltage output of the PEPS for each reservoir. During thepinched injection, 400 V was applied to the reservoir S for 30 s,280 and 600 V were applied to the reservoirs B and BW,respectively, while reservoir SW was grounded. Separation wasperformed by applying 2500 V to reservoir B, 1800 V to thereservoirs S and SW with reservoir BW grounded.All solutions were filtered through a 0.22 μm nylon syringe

filter before being added into the chip.Analysis of Fluorescent Products in Mitochondria.

Mitochondria in uptake buffer was diluted and incubated at 37°C in the presence of ascorbate oxidase, 2 μM DAF-FM, and 5μM DBZTC for 40 min. Then the mitochondrial solutionswere diluted 20 times in electrophoresis running buffer andmeasured according to the microchip electrophoresis proce-dure.

Statistical Analysis. Data were expressed as the mean ±standard deviation. All experiments were repeated three times,and the data were calculated with Microsoft Excel. Forsignificance testing, the Student’s t test was used (a p < 0.05is considered significant).



■ RESULTS AND DISCUSSION

Microchip Electrophoretic Optimization. Because of thesimilar fluorescence properties of their reaction products,DBZTC and DAF-FM were employed as labeling reagents forO2

−• and NO (Supporting Information, Figure S2), respec-tively. DBZTC which has been utilized to detect superoxide incell extracts using MCE−LIF methods in our previousworks,21,33 reacts with O2

−• in a 1:1 molar ratio and thederivatization efficiency is quantitative, but DAF-FM has beenseldom used for NO determination in conjunction with MCE−LIF methods, owing to the AA and DHA interferences.34 Inorder to achieve reliable determination for O2

−• and NO, theexperimental conditions were carefully optimized to obtain thebest sensitivity and selectivity for the targeted analytes understudy, especially those for NO.As shown in Figure 1, DAF-FM, newly prepared from DAF-

FM DA through hydrolyzation by esterase, exhibited low

background signal (5 μM, Figure 1). When the concentrationof DAF-FM was under 2 μM, no background fluorescent signalwas observed (2 μM, Figure 1). Therefore, subsequent NOdetermination was performed with 2 μM newly prepared DAF-FM. On the other hand, interference with DAF-FM assistedintracellular NO measurements28 by dehydroascorbic acid(DHA) and ascorbic acid (AA) could be reduced effectivelythrough conversion of AA to DHA by addition of ascorbateoxidase (AO), and Scheme 1 illustrates the solution ofeliminating interference of AA and DHA with NO detection.Microchip electrophoresis separation of negatively charged

DAF-FMT (fluorescent product of NO), DAF-FM-DHA(fluorescent product of DHA), and neutral DBO (fluorescentproduct of O2

−• with DBZTC) was then investigated to achievesimultaneous determination of O2

−• and NO. Four kinds of

buffers (i.e., HEPES, phosphate, Tris, and borate buffers) werethus employed to gain better separation of DBO, DAF-FMT,and DAF-FM-DHA. Considering the potential application ofthe method for biological analysis, a suitable medium of pH 7.4was selected, except that of borate buffer with pH 9.2. It wasfound that Tris buffer solution was the most suitable to obtain acompromise among a short analysis time, a higher columnefficiency, and a better resolution (Supporting Information,Table S1).The typical electrophoregram of DBO, DAF-FMT, and

DAF-FM-DHA is illustrated in Figure 2, with DBO, DAF-FM-

DHA, and DAF-FMT migration times of 41, 54, and 58 s underthe optimized separation conditions, respectively. Separationcan be completed within 1 min, indicating that simultaneousand rapid determination of O2

−• and NO can be realized.Analytical Characteristics of the Method. An inves-

tigation was performed to determine the linear range, limit ofdetection (LOD), and reproducibility, and the results areshown in Table 1. Under the optimized conditions, electro-kinetic pinched sampling operations were used for injection andpeak areas were used to acquire calibration lines. Thecalibration ranges obtained for DBO were 1.4 × 10−8 to 1.5× 10−7 M and 1.5 × 10−7 to 1.5 × 10−6 M, and those obtainedfor DAF-FMT were 3.6 × 10−9 to 1.5 × 10−7 M and 1.5 × 10−7

to 5.0 × 10−6 M. The LODs, calculated based on signals equalto 3 times the standard deviation of the background, were 4.3and 1.1 nM for DBO and DAF-FMT, respectively. The volumeof the injected sample plug was experimentally measured to be123 pL by visual checking using an inverted fluorescentmicroscope (DM-IL, Leica, Germany) and a charge-coupleddevice (CCD) camera (DFC300FX, Leica, Germany).22

Therefore, the mass LODs for DBO and DAF-FMT werecalculated to be 0.53 and 0.13 amol, respectively. Reproduci-bilities obtained from migration time and peak area measure-ments were studied by six injections of standard solutions of100 nM DBO or 50 nM DAF-FMT consecutively.

Abundance of DBO and DAF-FMT in MitochondriaIsolated from HepG2 Cells. To assess the applicability of theproposed MCE−LIF detection assay for monitoring O2

−• andNO simultaneously in real biological samples, the method was

Figure 1. Typical microchip electropherograms of fluorescent probe ofDAF-FM: bottom, 2 μM; top, 5 μM. Buffer: 50 mM Tris, pH 7.4.

Scheme 1. Eliminating AA/DHA Interference with NODetection

Figure 2. Electropherograms of standard solutions of fluorescentderivatives (from bottom to top): 100 nM DBO (a); DAF-FM-DHA(b); 50 nM DAF-FMT (c); DBO + DAF-FM-DHA + DAF-FMT (d).MCE conditions: running buffer, 40 mM Tris, pH 7.4; injection time,30 s; separation electric field, 500 V/cm; effective separation distance,30 mm.



first used to monitor the two species in mitochondria of HepG2cells. The typical electropherogram is shown in Figure 3. On

the basis of the migration times, the peaks corresponding toDBO and DAF-FMT were identified, which represented O2

−•

and NO, respectively. Although ascorbate oxidase was appliedto eliminate the AA inhibition on DAF-FM and NO reaction,the signal of DAF-FM-DHA was almost negligible (Figure 3),probably because of more reactivity of DAF-FM to NO than toDHA29 and because of the dilution of the mitochondrialsamples during preparation. Ground on the linear regressionequation and the peak area, the mitochondrial DBO and DAF-FMT contents were determined to be 0.84 ± 0.09 and 0.29 ±0.04 μM (n = 3; mitochondrial protein concentration, 0.5 mg/mL), respectively.The peak identification was further confirmed by depletion

experiments, and the analytical results are illustrated in Figure4. When Tiron was administered on mitochondria, the peak ofDBO disappeared while the peak area of DAF-FMT increasedslightly. Since O2

−• can react with NO to form ONOO−,reducing the O2

−• concentration by Tiron can reduce ONOO−

production and improve the bioavailability of NO under thecircumstances. When hemoglobin was administered onmitochondria, the peak of DAF-FMT disappeared as expected,but the peak area of DBO remained almost unchanged. Thereason for that might be due to the proportion of superoxidethat is directed toward peroxynitrite formation in the system. Ifthe majority of O2

−• reacts with targets rather than NO, theproduction of O2

−• will exceed that of NO. In this case, theadded hemoglobin is likely to decrease ONOO− formation withno effect on O2

−• production.35

Furthermore, nonrespiring mitochondria treated withrotenone were utilized as a control to confirm the biologicalsignificance of mitochondrial superoxide and nitric oxide

measurements by the proposed MCE−LIF method. Rotenoneis a respiratory inhibitor that blocks electron transfer throughcomplex I.36 Upon addition of rotenone, the DBO peak areaincreased by ∼2.7-fold (data not shown), a magnitude ofchange comparable with the 2−3-fold increment reported inliterature,37 indicating the superoxide release upon mitochon-drial electron transfer block. Meanwhile, the DAF-FMT peakarea had ∼0.45-fold decrease (data not shown), with thepossible reason that retonone could inactivate mitochondrialcomplex I, whereby complex I inactivation abolishes mtNOSactivity, thus reducing the NO amount accordingly.38

Measurements of DBO and DAF-FMT in IsolatedMitochondria from HepG2 Cells Undergoing Apoptosis.The proposed MCE−LIF assay was next applied to surveyvariation of DBO and DAF-FMT levels in mitochondria duringcell apoptosis. Resveratrol (trans-3,4′,5-trihydroxystilbene), apolyphenolic phytoalexin present mainly in grapes, red wine,and berries, was administrated on HepG2 cells to induceapoptosis. Resveratrol has been reported to have diversebeneficial actions, such as protecting cells and tissues againstneurodegeneration, cardiovascular disease, cancer, and diabetes.Moreover, it is also known to possess strong chemopreventiveand anticancer properties.39 Herein, the antiproliferative andapoptosis-inducing activities of resveratrol in human HepG2cells were investigated.Studies indicated that resveratrol induced apoptosis in

HepG2 cells in a dose- and time-dependent manner. Aconcentration of 100 μM resveratrol was administered onHepG2 cells according to the literature.40 The DAPI stainingfor chromatin condensation assessment demonstrated a nuclear

Table 1. Analytical Performance of the Proposed Method

analyte regression equationa R linear range (M)LOD(nM)b

RSD (%, n = 6)(migration time)

RSD (%, n = 6) (peakarea)

DBOc = ± + ±y x(0.049 0.17) (71.66 3.88) 0.9938 1.4 × 10−8−1.5 × 10−7 4.3 1.2 3.2

= ± + ±y x(2.86 1.43) (44.20 0.99) 0.9972 1.5 × 10−7−1.5 × 10−6

DAF-FMTd

= ± + ±y x(0.11 0.27) (270.91 8.44) 0.9980 3.6 × 10−9−1.5 × 10−7 1.1 1.8 4.1

= ± + ±y x(1.384 3.51) (247.31 10.52) 0.9991 1.5 × 10−7−5.0 × 10−6

ay, peak area (in mV·s); x, concentration of the analyte (in μM). bConcentration limits of detection measured for a signal/noise ratio of 3. Thestandard deviation of reagent blank is 0.103 (n = 10). cSpecies being detected: O2

−•. dSpecies being detected: NO.

Figure 3. Typical microchip electropherogram of fluorescent productsin mitochondria isolated from HepG2 cells. MCE conditions were thesame as in Figure 2.

Figure 4. Typical microchip electropherogram of DBO and DAF-FMT in mitochondria isolated from HepG2 cells (black line);electropherogram of mitochondrial fluorescent products of HepG2cells in the presence of hemoglobin (10 μM, green line) and Tiron (1mM, red line). MCE conditions were the same as in Figure 2.



marginalization within 6 h after resveratrol treatment (Figure5A) as an early sign of apoptosis. At the same time,

mitochondrial DBO and DAF-FMT amounts all increased(Figure 5B), indicating that resveratrol-induced apoptosisinvolved the participation of O2

−• and NO. It is interestingto note that the amounts of the two fluorescent products keptincreasing and that of DAF-FMT reached a peak of almost 2.5times versus control at 12 h of induction, partly because ofresveratrol modulation of mitochondrial NOS expression andactivation.41 From then on, there is also an increase in thenumber of apoptotic cells, which were suggested by cellshrinkage, chromatin condensation, nuclear fragmentation, andshedding (Figure 5A), after treatment of cells with resveratrolfor 18 and 24 h. The corresponding signal of DBO inmitochondria sustained a growing state and rose to 3 timesuntil 30 h disposal with resveratrol; however, mitochondrialDAF-FMT amount decreased until resveratrol treatment for 30h, suggesting that the activation of the NOS system might beonly an early event of apoptosis42 and the decrease in DAF-FMT might be due to NO reaction with O2

−• to form moredamaging ONOO−.5

Measurements of DBO and DAF-FMT in IsolatedMitochondria from PC12 Cells Undergoing Apoptosis.Increasing evidence suggests an important role of mitochon-drial dysfunction in the pathogenesis of Alzheimer’s disease, the

most common neurodegenerative disorder characterized by thepresence of amyloid plaques and programmed cell death.43,44

Thus, using the proposed MCE−LIF method, we investigatedthe effects of amyloidβ25−35 peptide (Aβ25−35) on mitochondrialO2

−• and NO production in PC12 cells, a useful in vitro modelfor neuronal differentiation. The typical electropherogram ofuntreated PC12 cells is shown in Figure 6, and the

mitochondrial DBO and DAF-FMT contents were 0.70 ±0.05 and 0.75 ± 0.11 μM (n = 3; mitochondrial proteinconcentration, 0.5 mg/mL), respectively. Excessive generationof DAF-FMT compared with liver cells manifested theimportant role of NO in signaling transduction in neuronalcells.45

It has been reported that Aβ can affect the dynamics ofmitochondria, a process which involves the participation ofROS and NO.46 Such a detailed work related to changes ofmitochondrial O2

−• and NO production has never been donebefore. Basal apoptosis of PC12 cells induced by 30 μM Aβ25−35was then observed using DAPI staining. It was found that 6 h ofincubation of PC12 cells with Aβ25−35 caused nuclearmorphological change (Figure 7A) plus DBO and DAF-FMTamount increase (Figure 7B), suggesting that O2

−• and NOmay be mediators of Aβ25−35-induced neuronal cell death. At 12h, several cells lose chromatin integrity (Figure 7A) and bothspecies keep increase to almost 2 times the level compared withcontrol samples. After 18 h, the amount of DAF-FMT reacheda platform and remained almost constant at 2.2 times incontrast to untreated mitochondria, but that of DBO showedsustained growth and featured a nearly 3.5-fold increase after 30h of induction with Aβ (Figure 7B). Simultaneously, the DAPIstaining demonstrated condensed chromatin gathering, nuclearfragmentation, and increased number of apoptotic cells,displaying the characteristics of apoptosis.In short, mitochondria serve as an important cellular

mediator of apoptosis, and different bioactive species such asROS and RNS may participate in the process. Our experimentalresults implied that O2

−• and NO were essential partners in thetwo cell types of apoptosis. Superoxide demonstratedunanimous increase, whereas nitric oxide showed a differenttrend during the process. This complexity may be aconsequence of the rate of NO production and the interactionwith other biological molecules such as O2

−•.

Figure 5. (A) DAPI staining of HepG2 cells upon 100 μM resveratroltreatment. (B) Time course of mitochondrial fluorescent productformation after resveratrol-induced HepG2 cell apoptosis. MCEconditions were the same as in Figure 2. Three independentmeasurements were carried out for each mitochondrial preparation.The error bar superimposed on each marker means SD.

Figure 6. Typical microchip electropherogram of DBO and DAF-FMT in mitochondria isolated from PC12 cells. MCE conditions werethe same as in Figure 2.



■ CONCLUSIONIn this work, the simultaneous determination of O2

−• and NOusing an MCE−LIF method has been established for the firsttime based on fluorescent probes DBZTC and DAF-FM. Thefeasibility of the assay was testified by measurement of the twoROS/RNS species in subcellular organelles. The tendency ofthe content change of the two species in mitochondria wasobtained during the course of apoptosis. The method is simple,fast, reproducible, and efficient. Furthermore, it provides apowerful tool to study the functions and interactions of the twospecies and new chances for understanding their pathobio-logical and therapeutic basis for diverse diseases.

■ ASSOCIATED CONTENT*S Supporting InformationAdditional information as noted in text. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected]. Fax: 86-531-86180017.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This work was supported by the National Key Natural ScienceFoundation of China (No. 21035003), the Specialized ResearchFund for the Doctoral Program of Higher Education of China(20113704130001), the National Natural Science Foundationof China (No. 21105057), the Key Natural Science Foundationof Shandong Province of China (No. ZR2010BZ001), and theProgram for Changjiang Scholars and Innovative ResearchTeam in University. The first two authors contributed equallyto this work.

■ REFERENCES(1) West, A. P.; Brodsky, I. E.; Rahner, C.; Woo, D. K.; Erdjument-Bromage, H.; Tempst, P.; Walsh, M. C.; Choi, Y.; Shadel, G. S.;Ghosh, S. Nature 2011, 472, 476−480.(2) Thomas, D. D.; Ridnour, L. A.; Isenberg, J. S.; Flores-Santana,W.; Switzer, C. H.; Donzelli, S.; Hussain, P.; Vecoli, C.; Paolocci, N.;Ambs, S.; Colton, C. A.; Harris, C. C.; Roberts, D. D.; Wink, D. A. FreeRadical Biol. Med. 2008, 45, 18−31.(3) Ghafourifar, P.; Schenk, U.; Klein, S. D.; Richter, C. J. Biol. Chem.1999, 274, 31185−31188.(4) Poderoso, J. J.; Lisdero, C.; Schopfer, F.; Riobo, N.; Carreras, M.C.; Cadenas, E.; Boveris, A. J. Biol. Chem. 1999, 274, 37709−37716.(5) Moncada, S.; Erusalimsky, J. D. Nat. Rev. Mol. Cell Biol. 2002, 3,214−220.(6) Deschacht, M.; Horemans, T.; Martinet, W.; Bult, H.; Maes, L.;Cos, P. Free Radical Res. 2010, 44, 763−772.(7) Amatore, C.; Arbault, S.; Bouton, C.; Drapier, J. C.; Ghandour,H.; Koh, A. C. ChemBioChem 2008, 9, 1472−1480.(8) Wegerich, F.; Turano, P.; Allegrozzi, M.; Mohwald, H.; Lisdat, F.Anal. Chem. 2009, 81, 2976−2984.(9) Yamaguchi, S.; Kishikawa, N.; Ohyama, K.; Ohba, Y.; Kohno, M.;Masuda, T.; Takadate, A.; Nakashima, K.; Kuroda, N. Anal. Chim. Acta2010, 665, 74−78.(10) Cornelius, J.; Tran, T.; Turner, N.; Piazza, A.; Mills, L.; Slack,R.; Hauser, S.; Alexander, J. S.; Grisham, M. B.; Feelisch, M.;Rodriguez, J. Biol. Chem. 2009, 390, 181−189.(11) Mukhopadhyay, P.; Rajesh, M.; Batkai, S.; Kashiwaya, Y.; Hasko,G.; Liaudet, L.; Szabo, C.; Pacher, P. Am. J. Physiol.: Heart Circ. Physiol.2009, 296, H1466−H1483.(12) Carroll, J. S.; Ku, C. J.; Karunarathne, W.; Spence, D. M. Anal.Chem. 2007, 79, 5133−5138.(13) Lebuffe, G.; Schumacker, P. T.; Shao, Z. H.; Anderson, T.;Iwase, H.; Vanden Hoek, T. L. Am. J. Physiol.: Heart Circ. Physiol.2003, 284, H299−H308.(14) Ling, Y. Y.; Yin, X. F.; Fang, Z. L. Electrophoresis 2005, 26,4759−4766.(15) Bliss, C. L.; McMullin, J. N.; Backhouse, C. J. Lab Chip 2007, 7,1280−1287.(16) Yang, W.; Sun, X.; Wang, H. Y.; Woolley, A. T. Anal. Chem.2009, 81, 8230−8235.(17) Reichmuth, D. S.; Wang, S. K.; Barrett, L. M.; Throckmorton,D. J.; Einfeld, W.; Singh, A. K. Lab Chip 2008, 8, 1319−1324.(18) Qin, J.; Ye, N.; Yu, L.; Liu, D.; Fung, Y.; Wang, W.; Ma, X.; Lin,B. Electrophoresis 2005, 26, 1155−1162.(19) Zhu, L.; Lu, M.; Yin, X. Talanta 2008, 75, 1227−1233.(20) Gao, J.; Yin, X. F.; Fang, Z. L. Lab Chip 2004, 4, 47−52.(21) Li, H.; Li, Q.; Wang, X.; Xu, K.; Chen, Z.; Gong, X.; Liu, X.;Tong, L.; Tang, B. Anal. Chem. 2009, 81, 2193−2198.(22) Chen, Z.; Li, Q.; Wang, X.; Wang, Z.; Zhang, R.; Yin, M.; Yin,L.; Xu, K.; Tang, B. Anal. Chem. 2010, 82, 2006−2012.(23) Brunati, A. M.; Pagano, M. A.; Bindoli, A.; Rigobello, M. P. FreeRadical Res. 2010, 44, 363−378.(24) Mishra, D. P.; Shaha, C. J. Biol. Chem. 2005, 280, 6181−6196.(25) Burwell, L. S.; Brookes, P. S. Antioxid. Redox Signaling 2008, 10,579−599.

Figure 7. (A) DAPI staining of PC12 cells upon 30 μM Aβ25−35treatment. (B) Time course of mitochondrial fluorescent productsformation after Aβ25−35-induced PC12 cell apoptosis. MCE conditionswere the same as in Figure 2. Three independent measurements werecarried out for each mitochondrial preparation. The error barsuperimposed on each marker means SD.



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(26) Gao, J. J.; Xu, K. H.; Tang, B.; Yin, L. L.; Yang, G. W.; An, L. G.FEBS J. 2007, 274, 1725−1733.(27) Zhang, X.; Kim, W. S.; Hatcher, N.; Potgieter, K.; Moroz, L. L.;Gillette, R.; Sweedler, J. V. J. Biol. Chem. 2002, 277, 48472−48478.(28) Kim, W. S.; Ye, X.; Rubakhin, S. S.; Sweedler, J. V. Anal. Chem.2006, 78, 1859−1865.(29) Ye, X.; Rubakhin, S. S.; Sweedler, J. V. Analyst 2008, 133, 423−433.(30) Carreras, M. C.; Poderoso, J. J. Am. J. Physiol.: Cell Physiol. 2007,292, C1569−C1580.(31) Liu, X.; Feng, L.; Yan, M.; Xu, K.; Yu, Y.; Zheng, X. Mol. Cell.Biochem. 2010, 344, 277−284.(32) Yang, Q.; Zhang, X.; Bao, X.; Lu, H.; Zhang, W.; Wu, W.; Miao,H.; Jiao, B. J. Chromatogr., A 2008, 1201, 120−127.(33) Liu, X.; Li, Q.; Gong, X.; Li, H.; Chen, Z.; Tong, L.; Tang, B.Electrophoresis 2009, 30, 1077−1083.(34) Nagata, N.; Momose, K.; Ishida, Y. J. Biochem. 1999, 125, 658−661.(35) Manser, R. C.; Houghton, F. D. J. Cell Sci. 2006, 119, 2048−2055.(36) Meany, D. L.; Thompson, L.; Arriaga, E. A. Anal. Chem. 2007,79, 4588−4594.(37) Xu, X.; Arriaga, E. A. Anal. Chem. 2010, 82, 6745−6750.(38) Parihar, M. S.; Nazarewicz, R. R.; Kincaid, E.; Bringold, U.;Ghafourifar, P. Biochem. Biophys. Res. Commun. 2008, 366, 23−28.(39) Notas, G.; Nifli, A. P.; Kampa, M.; Vercauteren, J.; Kouroumalis,E.; Castanas, E. Biochim. Biophys. Acta 2006, 1760, 1657−1666.(40) Ma, X.; Tian, X.; Huang, X.; Yan, F.; Qiao, D.Mol. Cell. Biochem.2007, 302, 99−109.(41) Finocchietto, P. V.; Franco, M. C.; Holod, S.; Gonzalez, A. S.;Converso, D. P.; Antico Arciuch, V. G.; Serra, M. P.; Poderoso, J. J.;Carreras, M. C. Exp. Biol. Med.(Maywood) 2009, 234, 1020−1028.(42) Hattori, R.; Otani, H.; Maulik, N.; Das, D. K. Am. J. Physiol.:Heart Circ. Physiol. 2002, 282, H1988−H1995.(43) Keil, U.; Bonert, A.; Marques, C. A.; Scherping, I.; Weyermann,J.; Strosznajder, J. B.; Muller-Spahn, F.; Haass, C.; Czech, C.; Pradier,L.; Muller, W. E.; Eckert, A. J. Biol. Chem. 2004, 279, 50310−50320.(44) Hardy, J.; Selkoe, D. J. Science 2002, 297, 353−356.(45) Nisoli, E.; Carruba, M. O. J. Cell Sci. 2006, 119, 2855−2862.(46) Kadowaki, H.; Nishitoh, H.; Urano, F.; Sadamitsu, C.;Matsuzawa, A.; Takeda, K.; Masutani, H.; Yodoi, J.; Urano, Y.;Nagano, T.; Ichijo, H. Cell Death Differ. 2005, 12, 19−24.



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